This application proposes a series of studies designed to determine the involvement of cyclic 3':5' adenosine monophosphate-(cAMP)-dependent protein kinase (cAMP-PK) in the hormone regulated construction and maturation of ovarian follicles. A recently established procedure that employs a fluorescinated conjugate of the heat stable inhibitor of free catalytic subunits disassociated from cAMP-PK has made possible the localization of those subunits in Reuber H-35 hepatoma cells. When stimulated with dibutyryl cAMP (DBcAMP) cAMP-PK was activated in a dose and time related manner and the disassociated catalytic subunits translocated to the nucleus over a period of time. It is proposed to apply this method to studies of granulosa cells harvested from ovarian follicles at discrete stages of differentiation. Cells will be stimulated with specific hormones or DBcAMP and the temporal and spatial kinetics of catalytic subunits freed from the holoenzyme as a consequence of these stimulations will be followed. The current method will be modified for use on frozen sections of tissues. Once validated this technique will be used to study cAMP-PK activation in vivo. These studies will be carried out with whole ovaries from animals pretreated with hormone or with intact, isolated follicles incubated for periods of times with specific stimuli. In both cases the activation of cAMP-PK will be determined and the kinetics of freed catalytic subunits followed. It is anticipated that these correlated in vivo and in vitro studies will provide insights at the cellular level, into some of the mechanisms involved in cAMP-pk mediated cell responses to hormones that regulates the construction and functional differentiation of ovarian follicles.